Pemphigus vulgaris (PV) is an autoimmune blistering disease of the skin and mucous membranes, which is histologically characterized by intraepidermal blister formation and immunopathologically characterized by the IgG autoantibody against the keratinocyte cell surface (Stanley, J. R. Pemphigus. In Dermatology in General Medicine. I. M. Freedberg, A. Z. Eisen, K. Wolff, K. F. Austen, L. A. Goldesmith, S. I. Katz, and T. B. Fitzpatrick, eds. McGraw-Hill, New York, 654-666 (1998)). Clinically, patients with pemphigus vulgaris exhibit extensive flaccid blister and erosion. These can occur in every stratified squamous epithelium. Pemphigus vulgaris can be lethal, since failing to conduct an appropriate therapy results in the induction of the leakage of body fluid or secondary bacterial infection caused by the focus developed at a wide range in the skin. The prognosis of pemphigus is being improved by systemic administration of corticosteroid and immunosuppression therapy, however, its mortality rate remains very high because of the death due to the complication of therapy.
The target antigen of pemphigus vulgaris was first identified by immunoprecipitation of keratinocyte extracts as a 130 kD glycoprotein (J. Clin. Invest. 70, 281-288, 1982; J. Clin. Invest. 74, 313-320, 1984). Then, cDNA of pemphigus vulgaris antigen was isolated by immunoscreening human keratinocyte expression libraries, with the use of an affinity-purified autoantibody specific to the pemphigus vulgaris antigen (Cell, 67, 869-877, 1991). Base sequence analysis showed that pemphigus vulgaris antigen belongs to the cadherin supergene family of cell-cell adhesion molecules. Pemphigus vulgaris antigen is a desmosomal transmembrane protein (J. Cell Biol. 122, 409-415, 1993) and was named desmoglein3 (Dsg3) (Adv. Dermatol. 11, 319-352, 1996).
There exist many evidences indicating that the IgG autoantibody against Dsg3 proteins have an aetiologic role for pemphigus vulgaris. Firstly, it is reported by indirect immunofluorescence (Br. J. Dermatol. 84, 7-13, 1971) or ELISA (J. Immunol. 159, 2010-2017, 1997; Br. J. Dermatol. 140, 351-357, 1999), that the chronological disease activity correlates with blood antibody titer. Secondly, the neonates of mothers with pemphigus vulgaris have transient disease due to maternal IgG that crosses through the placenta (Pediatrics 78, 1102-1105, 1986). As the maternal IgG is catabolized, the symptoms are relieved. Thirdly, the IgG derived from patients with pemphigus vulgaris, without complement or inflammatory cells, can induce blister formation in tissue-cultured skin (J. Invest. Dermatol. 67, 254-260, 1976; J. Exp. Med. 157, 259-272, 1983). Fourthly, passive transfer of IgG derived from patient sera to neonatal mice results in an intraepidermal blister formation with typical histological findings (N. Engl. J. Med. 306, 1189-1196, 1982). Fifthly, when the patient sera is removed by immunoabsorption with recombinant Dsg3 protein (rDsg3) which comprises an extracellular domain, the pathogenicity of the sera is removed, thereby inhibiting the blister formation in neonatal mice (J. Clin. Invest. 94, 59-67, 1994). Lastly, the antibody affinity-purified by rDsg3 has antigenicity, and forms a blister with histological findings of pemphigus vulgaris in neonatal mice (J. Clin. Invest. 90, 919-926, 1992; J. Clin. Invest. 102, 775-782, 1998). As can be seen from these studies, pemphigus vulgaris is one of the autoimmune diseases wherein its characteristic is most determined especially in the process after the production of autoantibody. Therefore, at present, pemphigus vulgaris is said to be an excellent disease model for organ-specific autoimmune disease, in order to study the cell mechanism of autoantibody production or breakdown of self-tolerance, and also to develop a disease-specific therapeutic method.
Based on the hypothesis that pathogenic antibodies are not produced in the body of mice due to the self-tolerance against Dsg3 proteins, the present inventors developed a method for generating mice that show phenotype of pemphigus vulgaris (PV mouse model), based on the finding that the immune system of Dsg3-deficient mice generated by gene targeting techniques is not exposed to Dsg3 proteins at the developmental stage, and therefore, self-tolerance against Dsg3 is not acquired (Japanese Patent Application Nos. H11-91408 and 2001-156126). That is, a method for generating mice that show phenotype of pemphigus vulgaris, comprised of the following steps: mice deficient in Dsg3 gene are immunized with recombinant Dsg3 protein (rDsg3 protein), splenocytes are prepared from the immunized mice, the splenocytes prepared are transferred to mice expressing Dsg3 proteins, and an antibody that reacts to Dsg3 proteins is produced or T cells are activated (Japanese Patent Application No. H11-91408), or a method for generating mice that show phenotype of pemphigus vulgaris, comprised of the following steps: splenocytes prepared from mice deficient in naive Dsg3 gene are prepared to 5×107 cells/mouse, the cells are transferred to the mice expressing Dsg3 proteins, and an antibody that reacts to Dsg3 proteins is produced or T cells are activated (Japanese Patent Application No. 2001-156126).
As mentioned above, pemphigus vulgaris is an autoimmune blistering disease against Dsg3, a cell-cell adhesion molecule, wherein the autoantibody having a pathogenicity involved in its onset is polyclonal, and the pathogenicity of their individual autoantibody is unknown. The PV model mice developed by the present inventors mentioned above are induced with its phenotype by anti-Dsg3 antibody having the pathogenicity. Therefore, it is considered that generating an anti-Dsg3 monoclonal antibody and analyzing the pathogenicity in the individual monoclonal antibodies are useful for the elucidation of the onset mechanism of pemphigus vulgaris at the molecular level, and for the development of antigen-specific antibody elimination therapy. However, to the present, there are no reports of a monoclonal antibody having a pathogenicity that can induce pemphigus vulgaris lesion. The object of the present invention is to provide a monoclonal antibody having a pathogenic activity that can induce pemphigus vulgaris lesion that is thought to be useful for the elucidation of the onset mechanism of pemphigus vulgaris at the molecular level, and for the development of antigen-specific antibody elimination therapy, a peptide specifically recognized by the monoclonal antibody thought to be useful as a therapeutic drug for pemphigus autoimmune disease that, etc.